Integrated sample processing for electrospray ionization devices

ABSTRACT

Methods, systems and devices that generate differential axial transport in a fluidic device having at least one fluidic sample separation flow channel and at least one ESI emitter in communication with the at least one sample separation flow channel. In response to the generated differential axial transport, the at least one target analyte contained in a sample reservoir in communication with the sample separation channel is selectively transported to the at least one ESI emitter while inhibiting transport of contaminant materials contained in the sample reservoir toward the at least one ESI emitter thereby preferentially directing analyte molecules out of the at least one ESI emitter. The methods, systems and devices are particularly suitable for use with a mass spectrometer.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/285,186, filed Oct. 4, 2016 (allowed), which is a continuation ofU.S. patent application Ser. No. 14/402,278, filed Nov. 19, 2014, nowU.S. Pat. No. 9,502,225, issued Nov. 22, 2016, which is a §371 NationalStage Patent Application of PCT Application Serial NumberPCT/US2013/044266, filed Jun. 5, 2013, which claims priority to and thebenefit of U.S. Provisional Application Ser. No. 61/662,152, filed Jun.20, 2012, the contents of which are hereby incorporated by reference asif recited in full herein.

FIELD OF THE INVENTION

This invention is related to electrospray ionization devices that areparticularly suitable for infusion-based devices that interface withmass spectrometers.

BACKGROUND OF THE INVENTION

Electrospray ionization (“ESI”) is an important technique for theanalysis of biological materials contained in solution by massspectrometry. See, e.g., Cole, R. B. Electrospray Ionization MassSpectrometry: Fundamentals, Instrumentation & Applications; John Wileyand Sons, Inc.: New York, 1997. Electrospray ionization was developed inthe late 1980s and was popularized by the work of Fenn. See, e.g., FennJ B, Mann M, Meng C K, Wong S F & Whitehouse CM (1989), Electrosprayionization for mass-spectrometry of large biomolecules. Science 246,64-71. Simplistically, electrospray ionization involves the use ofelectric fields to disperse a sample solution into charged droplets.Through subsequent evaporation of the droplets, analyte ions containedin the droplet are either field emitted from the droplet surface or theions are desolvated resulting in gas phase analyte ions. The source ofthe liquid exposed to the electric field and to be dispersed is ideallyone of small areal extent as the size of the electrospray emitterdirectly influences the size of droplets produced. Smaller dropletsdesolvate more rapidly and have fewer molecules present per dropletleading to greater ionization efficiencies. These ions can becharacterized by a mass analyzer to determine the mass-to-charge ratio.Further analyte structural information can be obtained by employingtandem mass spectrometry techniques.

The chemical informing power of electrospray ionization—massspectrometry can be enhanced when the electrospray emitter is coupled toliquid-phase chemical separations such as liquid chromatography,capillary electrophoresis, or ion exchange chromatography, to name afew. These chemical separation techniques endeavor to deliver isolatedcompounds to the electrospray emitter to reduce ionization suppressionand mass spectral complexity.

When performing electrospray ionization-mass spectrometry (ESI-MS) it isoften necessary to first remove unwanted components of the samplematrix. These unwanted components can cause a number of problems forESI-MS including: ionization suppression, complexation with analyteions, and fouling of the mass spectrometer inlet. For large molecules,like intact proteins that generate complex mass spectra, common samplematrix components like surfactants and buffer salts can render the massspectrum unintelligible. Furthermore, it is often difficult and timeconsuming to completely remove these unwanted components by conventionalmethods.

SUMMARY OF EMBODIMENTS OF THE INVENTION

Embodiments of the invention provide systems, methods and devicesconfigured to generate online sample processing using differential axialtransport between analytes of interest and contaminate materials.

Embodiments of the invention are directed to methods for online sampleprocessing for electrospray ionization (which may be particularly usefulfor mass spectrometry). The methods include: (a) generating differentialaxial transport in a fluidic device having at least one fluidic sampleseparation flow channel and at least one ESI emitter in communicationwith the at least one sample separation flow channel; and (b) inresponse to the generated differential axial transport, selectivelytransporting at least one target analyte contained in a sample reservoirin communication with the sample separation flow channel to the ESIemitter while inhibiting transport of contaminant materials contained inthe sample reservoir to the ESI emitter. The analyte molecules thusbeing preferentially directed out of the ESI emitter.

The at least one target analyte can include a protein and the at leastone non-analyte component can include a surfactant.

The fluidic device can have at least one background electrolyte (BGE)flow channel, a respective BGE channel merging into a respectiveseparation channel at a junction proximate the at least one emitter.Generating the differential axial transport can include: (i) applying afirst voltage proximate an ingress end portion of the separationchannel; (ii) concurrently applying a second different voltage proximatean ingress end portion of the BGE channel; and (iii) generating axialEOF in the separation and BGE channels in response to the application ofthe voltages so that the EOF in the BGE channel has a greater mobilitythan that in the separation channel.

The fluidic device can have at least one sample reservoir that feeds thesample separation flow channel. The fluidic device can include at leastone BGE reservoir and associated BGE channel in communication with theat least one sample separation flow channel. Generating the differentialaxial transport and the selective transport can be carried out by: (i)applying a first voltage to the sample reservoir; (ii) applying a secondvoltage to the BGE reservoir; and (iii) generating an EOF in the sampleseparation flow channel that is toward the sample reservoir and awayfrom the ESI emitter; and (iv) generating an EOF in the BGE channel,with the EOF flow in the BGE channel being greater than the EOF flow inthe separation channel.

In particular embodiments, the analyte can have an electrophoreticvelocity that is above the EOF of the separation channel and thenon-analyte component can have less mobility than the target analytesuch that there is selective transport of the at least one targetanalyte from the sample reservoir against the EOF in the separationchannel toward the at least one ESI emitter and the non-analyte remainsin the sample reservoir.

The selective transport can be carried out by allowing high mobilitycationic analyte molecules to migrate against the EOF in the separationchannel from a sample reservoir toward the ESI emitter while lowmobility cations, anions, and neutrals remain in the sample reservoir.

The selective transport can be carried out by causing high mobilityanionic analyte molecules to migrate against the EOF in the separationchannel toward the ESI emitter while low mobility anions, cations, andneutrals remain in a sample reservoir holding a supply of the sample,the sample reservoir being in fluid communication with the separationchannel.

The EOF in the BGE channel can be greater than that in the separationchannel and can define an EO pump that drives fluid with the at leastone analyte out of the at least one emitter to spray toward a collectiondevice for subsequent analysis and/or toward an inlet of a massspectrometer.

The fluidic device can be a capillary device with the separation channeldefined by a capillary tube.

The fluidic device can be a microfluidic chip.

The method can include spraying fluid with the analyte out the at leastone emitter in a substantially stable stream or plume for periods oftime sufficient for generating signal averaged mass spectra, ionmobility spectra, or for collection of the analyte onto a plate forMALDI analysis of the analyte.

The at least one target analyte can include intact monoclonalantibodies.

Other embodiments are directed to mass spectrometer analyzer systemswith integrated online sample processing (e.g., cleaning). The systemsinclude: (a) a mass spectrometer with an inlet orifice; (b) a fluidicdevice having at least one sample reservoir, at least one fluidic sampleseparation flow channel that is in fluid communication with a respectivesample reservoir, at least one background electrolyte reservoir with anassociated (BGE) flow channel, and at least one ESI emitter incommunication with the at least one sample separation flow channel,wherein a respective BGE flow channel merges into a respectiveseparation channel at a junction proximate the at least one emitter; and(c) at least one power source having a first voltage input incommunication with the sample reservoir and a second voltage input incommunication with the BGE reservoir, wherein, in operation, the firstand second voltage inputs are both (e.g., substantially constantly)powered with one of the first and second voltages being greater than theother to generate axial electroosmotic flow (EOF) in the separation andBGE channels so that the EOF in the BGE channel has a greater mobilitythan that in the separation channel.

Optionally, the EOF mobility in the separation channel can be less thanan electrophoresis of the at least one target analyte. The fluidicdevice can include a dirty sample in the sample reservoir. The at leastone target analyte can migrate against the EOF in the separation channelto be emitted by the emitter while at least one unwanted non-analyteremains in the separation channel due to electrophoretic mobility biasto thereby provide integrated online sample cleaning.

The magnitude and direction of the EOF can be dictated by the channelsurface chemistry.

The system can include a control circuit configured to controlapplication of the voltages to generate the EOF.

Surfaces of the at least one separation channel can have a negativecharge (cathodic EOF). In operation, the at least one separation channelcan be configured to allow high mobility anions of the at least onetarget analyte to migrate against the EOF in the separation channel fromthe sample reservoir toward the ESI emitter while low mobility anions,cations, and neutrals remain in the sample reservoir.

Surfaces of the at least one separation channel can have a positivecharge (anodic EOF). In operation, the at least one separation channelcan be configured to allow high mobility cations of the analyte tomigrate against the EOF in the separation channel toward the ESI emitterwhile low mobility cations, anions, and neutrals remain in the samplereservoir.

Surfaces of the at least one separation channel can have a negativecharge. In operation, the at least one separation channel can beconfigured to allow the at least one target analyte to migrate with theEOF in the separation channel from the sample reservoir toward the ESIemitter.

Surfaces of the at least one separation channel can have a positivecharge. In operation, the at least one separation channel can beconfigured to allow the analyte to migrate with the EOF in theseparation channel toward the ESI emitter.

Other embodiments are directed to microfluidic devices forinfusion-electrospray ionization-mass spectroscopy. The devices include:(a) a background electrolyte (BGE) reservoir; (b) a BGE channel in fluidcommunication with the BGE reservoir; (c) a sample reservoir; (d) aseparation channel in fluid communication with the sample reservoir, theseparation channel having a defined positive or negative charge and amodified electroosmotic flow surface coating that reduces EOF flow alongan entire length thereof from the sample reservoir to a junction definedby an intersection of the BGE channel with the separation channel; and(e) at least one emitter in fluid communication with the separationchannel and the BGE channel proximate to but downstream of the junction.

The BGE reservoir, BGE channel, sample reservoir, separation channel andat least one emitter can be held on a microchip.

It is noted that aspects of the invention described with respect to oneembodiment, may be incorporated in a different embodiment although notspecifically described relative thereto. That is, all embodiments and/orfeatures of any embodiment can be combined in any way and/orcombination. Applicant reserves the right to change any originally filedclaim and/or file any new claim accordingly, including the right to beable to amend any originally filed claim to depend from and/orincorporate any feature of any other claim or claims although notoriginally claimed in that manner These and other objects and/or aspectsof the present invention are explained in detail in the specificationset forth below. Further features, advantages and details of the presentinvention will be appreciated by those of ordinary skill in the art froma reading of the figures and the detailed description of the preferredembodiments that follow, such description being merely illustrative ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic illustration of an exemplary fluidic deviceaccording to embodiments of the present invention.

FIG. 1B is a schematic illustration of another exemplary fluidic devicesimilar to that shown in FIG. 1A, but with a different electric inputfor the electric field according to embodiments of the presentinvention.

FIG. 2 is a schematic illustration of a fluidic device similar to thatshown in FIG. 1 but with reverse EOF electrical charge configurationsaccording to some embodiments of the present invention.

FIG. 3A is a schematic illustration of a microfluidic device accordingto embodiments of the present invention.

FIG. 3B is a schematic illustration of another microfluidic deviceaccording to embodiments of the present invention.

FIGS. 4A-4C are schematic illustrations of examples of capillary ESIsystems with integrated online sample processing according toembodiments of the present invention.

FIG. 5A is a top view of an example of a microfluidic device with onlineintegrated sample processing (e.g., cleanup) capability according toother embodiments of the present invention.

FIG. 5B is a (long) side view and FIG. 5C is an end view (e.g., shortside) of the microfluidic device shown in FIG. 5A according toembodiments of the present invention.

FIG. 6 is a block diagram of a system which includes at least onefluidic device configured for online sample processing usingdifferential axial transport in cooperating communication with a massspectrometer according to embodiments of the present invention.

FIG. 7 is a flow chart of exemplary operations that can be used to carryout embodiments of the present invention.

FIG. 8 is a schematic illustration of a prior art syringe pumpinfusion-ESI system used to generate data shown in FIGS. 9A and 9B.

FIGS. 9A and 9B are graphs of spectra, intensity (percentage) versus m/zof about 5 minutes of summed mass spectra associated with the deviceshown in FIG. 8.

FIGS. 10A and 10B are graphs of spectra, intensity (percentage) versusm/z of about 5 minutes of summed mass spectra associated with the deviceshown in FIG. 3A having integrated online sample clean up according toembodiments of the present invention.

FIGS. 11-16 are graphs of additional experimental data obtained usingdifferential axial transport between a target analyte and contaminatematerials according to embodiments of the present invention.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention will now be described more fully hereinafter withreference to the accompanying figures, in which embodiments of theinvention are shown. This invention may, however, be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth herein. Like numbers refer to like elementsthroughout. In the figures, certain layers, components or features maybe exaggerated for clarity, and broken lines illustrate optionalfeatures or operations unless specified otherwise. In addition, thesequence of operations (or steps) is not limited to the order presentedin the figures and/or claims unless specifically indicated otherwise. Inthe drawings, the thickness of lines, layers, features, componentsand/or regions may be exaggerated for clarity and broken linesillustrate optional features or operations, unless specified otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms, “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises,”“comprising,” “includes,” and/or “including” when used in thisspecification, specify the presence of stated features, regions, steps,operations, elements, and/or components, but do not preclude thepresence or addition of one or more other features, regions, steps,operations, elements, components, and/or groups thereof. As used herein,the term “and/or” includes any and all combinations of one or more ofthe associated listed items. As used herein, phrases such as “between Xand Y” and “between about X and Y” should be interpreted to include Xand Y. As used herein, phrases such as “between about X and Y” mean“between about X and about Y.” As used herein, phrases such as “fromabout X to Y” mean “from about X to about Y.”

It will be understood that when a feature, such as a layer, region orsubstrate, is referred to as being “on” another feature or element, itcan be directly on the other feature or element or intervening featuresand/or elements may also be present. In contrast, when an element isreferred to as being “directly on” another feature or element, there areno intervening elements present. It will also be understood that, when afeature or element is referred to as being “connected”, “attached” or“coupled” to another feature or element, it can be directly connected,attached or coupled to the other element or intervening elements may bepresent. In contrast, when a feature or element is referred to as being“directly connected”, “directly attached” or “directly coupled” toanother element, there are no intervening elements present. Althoughdescribed or shown with respect to one embodiment, the features sodescribed or shown can apply to other embodiments.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the present applicationand relevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. Well-known functions orconstructions may not be described in detail for brevity and/or clarity.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if the device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

It will be understood that, although the terms first, second, etc. maybe used herein to describe various elements, components, regions, layersand/or sections, these elements, components, regions, layers and/orsections should not be limited by these terms. These terms are only usedto distinguish one element, component, region, layer or section fromanother region, layer or section. Thus, a first element, component,region, layer or section discussed below could be termed a secondelement, component, region, layer or section without departing from theteachings of the present invention.

The term “about” means that the stated number can vary from that valueby +/−20%.

The term “analyte” refers to a molecule or substance undergoinganalysis, typically, at least for mass spectrometry analysis, having anion or ions of interest in a m/z range of interest. The analyte cancomprise biomolecules such as polymers, peptides, proteins and the like.Embodiments of the invention are particularly suitable for analyzingintact monoclonal antibodies.

The term “online sample processing” refers to separation of one or morenon-analyte sample constituents (e.g., contaminant materials) from oneor more target analytes in a respective sample during electrosprayemission of fluid with the analyte(s) undergoing analysis. Unwantednon-analyte sample constituents (e.g., contaminant materials) caninclude impurities such as surfactants and/or buffers that mightotherwise result in ESI-MS spectra that occludes or interferes with thegeneration of mass spectra, or other measurement information, of theanalyte itself. The unwanted non-analyte(s) can have an ion or ions ofinterest in an m/z range of interest that may overlap that of one ormore target analytes. Similarly, the term “dirty sample” refers to asample that has a target analyte and other unwanted non-analytes asnoted above. Thus, a dirty sample is a sample that has not beenpre-cleaned, filtered or processed to remove unwanted components.

The term “differential axial transport” means the use of hydraulictransport configured so that net transport of the analyte(s) is towardone or more ESI emitters without the contaminants in the sample/samplereservoir. The differential axial transport can include any combinationof EOF (magnitude and direction), electrophoretic mobility (magnitudeand direction) used with or without pressure-driven flow.

The term “microchip” refers to a substantially planar, thin, and, insome embodiments, rigid device. The term “thin” refers to a thicknessdimension that is less than about 10 mm, typically about 1 mm or less.The microchip typically has a width and length that is less than about 6inches and a thickness that is less than about 5 mm, typically betweenabout 2000 μm to about 250 μm.

The terms “integrated” and “integral” and derivatives thereof means thatthe component or process is incorporated into or carried out by afluidic device.

The term “high voltage” refers to voltage in the kV range, typicallybetween about 1-100 kV, more typically between about 1-20 kV. Lowervoltages may be used for precessing and/or in certain embodiments, ESIprocesses can employ potentials of a few kVs.

The term “microfluidic” refers to fluid flow channels that havesub-millimeter or smaller size width and/or depth (e.g., the termincludes nanometer size channels) and includes channels with width ordepth in a size range of about tens to hundreds of microns.

All of the document references (patents, patent applications andarticles) are hereby incorporated by reference as if recited in fullherein.

In typical free zone capillary electrophoresis (CE) experiments, asample plug is injected into a column, and an applied electric fieldcauses sample components to separate by differences in their mobilities.The mobility of a molecule is the sum of its electrophoretic mobilityand the electroosmotic mobility, and any pressure driven flow, ifpresent, of the separation column.

When the electroosmotic mobility is greater than the electrophoreticmobility of each sample component (e.g., constituent), all componentsmigrate in the same direction. However, when the electroosmotic mobilityis low, species with a higher electrophoretic mobility of opposite sign(polarity or charge) will migrate against the electroosmotic flow (EOF).Generally stated, embodiments of the invention use the latter propertyfor online sample processing to separate analyte molecules from samplematrix components, if the analyte molecules have sufficiently differentelectrophoretic mobilities than the other components. If the (CE)separation channel has a low electroosmotic mobility, analyte moleculeswith sufficiently high electrophoretic mobility can be constantly drawnout of a sample matrix by the application of an appropriate electricfield. Sample components that do not have sufficient mobility willremain in the sample reservoir. With an appropriate ESI interface,analyte molecules migrating down the separation column or channel can beconstantly sprayed or infused into a mass spectrometer, or into or ontoother devices.

The total mobility (μ or “mobility”) of a molecule can define itsvelocity (ν) in an electric field (E):

ν₁=μ₁E  Equation (1)

The total mobility of a molecule is the sum of its electrophoreticmobility (μ_(EP)) and the electroosmotic mobility (μ_(EO)) of atransport channel.

μ₁=μ_(EP1)+μ_(EO)  Equation (2)

If pressure-driven flow is also present in the transport channel, thenthe pressure-driven flow can either diminish or enhance the bulkelectroosmotic flow. Because pressure-driven flow is independent fromthe electric field, Equation (1) can be modified to include apressure-driven velocity (ν_(P)) component or term to modify theobserved velocity in the presence of pressure-driven flow per Equation(3).

ν₁=μ₁ E+ν _(P)  Equation (3)

To facilitate differential axial transport, the observed velocity of atarget analyte (ν₁) should be toward the ESI emitter or detector end ofthe transport (e.g., separation) channel, while the observed velocity orvelocities of the contaminant(s) or impurities (ν_(i)) can be in theopposite direction. If ν₁ is positive, then ν_(i) is negative and viceversa. As long as the electrophoretic mobilities μ₁ and μ_(i) aresufficiently different, conditions can be engineered to yield ν₁ andμ_(i) values of opposite sign. Therefore, the electrophoretic mobilityof the analyte is not always required to be (but can be) greater thanthat of the contaminants or impurities in a respective sample. Inaddition, the electrophoretic mobility of the analyte is not required tobe opposite that of the contaminant or impurities. The analyte canmigrate against the EOF as discussed herein in some embodiments.However, in some embodiments, the impurities can migrate against theEOF, and the analyte can migrate with the EOF. Referring to FIG. 1A,embodiments of the invention provide a fluidic device 10 (which can be amicrofluidic chip device) with at least one sample separation flowcolumn or channel 12 and at least one ESI interface 25, which istypically including a junction J of the separation channel 12 and abackground electrolyte channel 22 proximate at least one ESI emitter 15.The analyte molecules “A” have sufficiently greater cationicelectrophoretic mobilities than at least some of the other unwantedcomponents of the sample. The separation column 12 is configured to havea low electroosmotic mobility so that analyte molecules “A” that have agreater cationic electrophoretic mobility can be constantly drawn out ofa sample matrix by applying an electric field using electric inputs 116,120 for applying a defined voltage, which may be high voltages (HV)e.g., HV1, HV2, for example. However, other lower voltages or electricfield configurations can be used. A single sample (e.g., plug) in thesample reservoir 16 can be configured to generate a sufficient sprayduration, such as, by way of example only, between about 2 minutes toseveral hours, typically at least 5 minutes of substantially stablespray or output from a respective emitter to a mass spectrometer togenerate (summed) spectra data using a mass spectrometer. FIG. 1Billustrates that the electrical input 120 can be an electrical ground.

The devices 10, 10′ (FIGS. 4A-4C) described herein are particularlyuseful for infusion-ESI to provide the integrated sample processing. InFIGS. 1A and 2, the arrows indicate the direction and relative magnitudeof electroosmotic flow (EOF). In FIG. 1A, the dashed arrow indicates thedirection that a highly mobile cation would migrate with a positivesurface charge (anodic EOF)configuration of the channels 12, 22. Thereverse applies to the embodiment of FIG. 2. The ESI interface 25 can belocated at the far right end of the channel 12. The device 10 caninclude a sample reservoir 16 and a pump or background electrolyte (BGE)reservoir 20 with an associated channel 22. The at least one emitter 15can generate a substantially constant spray plume 15 p (FIG. 3A) orotherwise infuse the analyte “A” for collection onto a device forsubsequent analysis or directly into an inlet region of a massspectrometer 75 for generating spectra. The embodiments shown in FIGS.1A, 1B use positive charge to generate the electrospray of the analyte Afrom the emitter(s) 15. The surfaces of the flow channels 12, 22 canhave a positive charge “P” that can be provided by selection of thesubstrate material and/or coatings applied thereto.

The differential axial transport can be implemented with a microfluidicdevice (FIGS. 1-3) or with capillary tubing and connectors, FIGS. 4A-4C.

FIG. 2 illustrates an embodiment similar to FIG. 1A, but with an anionicsurface charge used to selectively transport the analyte A. In thisembodiment, the channels 12, 22 can be formed of or include a coatingthat has a negative charge “N” and the analyte A may have a negativecharge.

The sample reservoir 16 may be on the device 10 as shown or may resideupstream of the device 10 and be in fluid communication with the atleast one separation channel 12. The BGE reservoir 20 can reside on thedevice or reside upstream of the device and be in fluid communicationwith the respective channel 22. The junction (J) may reside proximatethe at least one emitter 15 on an outer edge or end of the device,typically between about 1-600 microns to reduce dead space/volumes.

FIGS. 1A and 1B are a simplified schematic of a device 10 with channels12, 22. FIGS. 1A, 1B illustrate both of the channels 12, 22 with thepositive charge coating 80 and channel 12 only with the reduced EOFcoating 88. When an electric field is applied to this fluidic network,highly mobile cations of analyte “A” migrate or travel against the EOFin the separation channel 12 as indicated by the dashed arrow over thechannel 12 toward the ESI interface 25. Low mobility cations, neutrals,and anions present in the sample matrix are excluded from the separationchannel 12 (or at least from substantial movement through the channel)and typically remain in the sample reservoir 16.

The mismatch in EOF between the BGE or pump channel 22 and theseparation channel 12 can, in some embodiments, also define anelectroosmotic pump which drives fluid out of the device 10 through theat least one emitter 15 for ESI-MS.

In some embodiments, the sample separation channel 12 can have anelectrical input 116 which is a voltage that is greater than the pump orBGE reservoir electrical input 120 (with both being positive or with thepump or BGE reservoir at ground). In other embodiments, the 116 voltagecan be less than the input 120 (and both may be positive). The selectedvoltage differential can depend on the analyte being transported, thetype of fluidic structure used to provide the flow, lengths, shapesand/or positions of the channels 12, 22 and the like.

One of the electric potentials 116, 120 applied can be a low voltage oreven an electrical ground. The inputs 116, 120 can be selected togenerate an electric field of proper magnitude and polarity for analytemigration and a suitable electrospray voltage. The magnitude of theelectric field strength can depend on the application and microfluidicdevice. In the absence of pressure-driven flow, the electric field candetermine the analyte migration velocity and the rate of bulk flow forsupporting the electrospray plume. Pressure-driven flow can enhance orcounter-balance either or both of these flows.

For capillary electrophoresis, typical field strengths usually rangefrom about 100 V/cm to well over 1000 V/cm. For positive ESI, theelectrospray voltage is usually between +1 and +5 kV, relative to thepotential of the mass spectrometer inlet. On some instruments the massspectrometer inlet 75 i has a voltage that can be varied, while on otherinstruments it remains fixed near ground.

In some particular embodiments, the voltage differential between theinput 116 and input 120 can be at least 20%, and even at least 50%greater. The electric inputs 116, 120 for applying the electric fieldcan be located at other positions than those shown, such as proximatethe respective reservoirs 16, 20 but upstream or downstream a distancethereof but in a position that applies the appropriate field and formssuitable EOFs.

The BGE or pump reservoir 20 can hold any suitable backgroundelectrolyte fluid appropriate for the target analyte(s) A andelectrospray output as is known to those of skill in the art. Forpositive biased devices/systems such as shown in FIGS. 1A, 1B a weakacid may be used such as an acetonitrile and formic acid solution.However, other background electrolyte fluids can be used as is wellknown. For a negative biased system/device such as shown in FIG. 2, abasic pH electrolyte can be used.

The EO pump 22 for electrospray ionization can take forms other thanshown in FIGS. 1-3. The EO pump can be described as the side channel 22with appropriately designed surface chemistry to create a desired EOflow (with reservoir 20 forming a pump reservoir). The basic requirementis to have two channels intersect at a junction (“J”), which may be aT-like or V-like junction (not restricted to a right angleintersection). Voltage is applied to two of the three resulting channeltermini generating an axial electric field through the associatedchannel segments. To realize hydraulic transport through the thirdchannel segment, the electroosmotic mobility in the two channel segmentsthat contain the axial electric field have a different magnitude.

The difference in electroosmotic mobility can be achieved by chemicallymodifying one, or both, of the associated channel segments so as toproduce different surface charge densities and hence differentelectroosmotic mobilities. Electroosmotic mobility can also be modifiedby coating a channel wall with electrically neutral polymer films,thereby increasing the effective fluid viscosity within the electricaldouble layer at the wall. Another way to modify electroosmotic mobilityis reduce one of the channel lateral dimensions to distances similar inmagnitude to the Debye length of the solution being electroosmoticallypumped. The described methods for modifying electroosmotic mobility mayalso be used in combination, where desired. Exemplary methods forelectroosmotic pumping are further described in U.S. Pat. No. 6,110,343,the contents of which are hereby incorporated by reference as if recitedin full herein.

For positive mobility bias embodiments, such as shown in FIGS. 1A, 1B atleast the surfaces of the separation channel and BGE or pump channel 12,22, respectively, can be configured with a suitable substrate and/orcovalently modified with a suitable coating 80 to provide a desiredpositive charge “P” as will be discussed further below. The coating 80or channel substrate material of each channel 12, 22 can be the same(typically the same and provided as a monolayer) or different to providea strong positive charge on all of the channel surfaces and therefore astrong anodic EOF.

For the negative bias embodiments, such as shown in FIG. 2, at least thesurfaces of the separation and BGE or pump channel 12, 22 can beconfigured with a negative charge based on the substrate material usedand/or a coating applied thereto for a strong cathodic EOF. Again, thesame material or coating(s) may be used or a different substratematerial and/or coating can be used to generate the negative charge. Forexample, a glass substrate defining the channels 12, 22 can provide anegative surface charge N.

Surface charge and ion migration direction can be such that if thesurface charge is negative (cathodic EOF), cations will migrate in thesame direction as the EOF. If the surface charge is positive (anodicEOF), anions will migrate in the same direction as the EOF.

The separation channel 12 can be modified to reduce EOF relative to theother channel 22 by a desired amount using an EOF reduction coatingmaterial 88 such as a viscous polymer. The differential in EOF cantypically be at least about 20% but the surfaces of the channels 12, 22can be configured to provide less or more EOF differential. As shown inFIGS. 1A and 2, in a relative measure, the channel 22 can have an EOF of(−10/+10) while the channel 12 can have an EOF of about (−0.8/+0.8).However, the EOF differential can vary depending on a number of factors

FIG. 3A is a schematic illustration of another embodiment of theinvention. The device 10 can be a microfluidic chip 10 c formed withstacked hard and/or soft substrates 11 (FIGS. 5A-5C) with integratedmicrofluidic channels 12, 22.

For CE separations, the cross structure shown in FIG. 3A is typicallyused for electrokinetically-gating sample plugs into the separationchannel For infusion-ESI methods or uses, such as that contemplated byembodiments of the invention, the cross structure is not necessary andthe reservoirs labeled BGE and waste in FIG. 3A are not used and/or maybe omitted from the device 10.

FIG. 3B illustrates another embodiment of the device 10 which can beimplemented as a fluidic microchip 10 c. The separation channel 12 canbe curvilinear with straight segments that reach from one short side ofthe chip to another where the emitter 15 is located so that the plume 15p of the analyte for the ESI output toward an MS inlet 75 i, accordingto some embodiments of the invention. As with FIGS. 1A, 1B and 2, FIGS.3A and 3B show that the device 10 can include at least one emitter 15,at least one separation channel 12 and at least one pump channel 20.Examples of hard or substantially rigid materials include, but are notlimited to, substrates comprising one or combinations of: glass, quartz,silicon, ceramic, silicon nitride, polycarbonate, andpolymethylmethacrylate. In particular embodiments, the device 10 caninclude a glass substrate such as a borosilicate. In other embodiments,a rigid polymer material may be used to form the microchip. The device10 can also include one or more layers of a soft or flexible substrate.Soft substrate materials, where used, can have a low Young's Modulusvalue. For example, elastomers and harder plastics and/or polymers canhave a range between about 0.1-3000 MPa. Examples of soft materialsinclude, but are not limited to, polydimethylsiloxane (PDMS),polymethylmethacrylate (PMMA), and polyurethane. See, e.g., co-pendingPCT/US2012/027662 filed Mar. 5, 2012 and PCT/US2011/052127 filed Sep.19, 2011 for a description of examples of microfabricated fluidicdevices. See, also. Mellors, J. S.; Gorbounov, V.; Ramsey, R. S.;Ramsey, J. M., Fully integrated glass microfluidic device for performinghigh-efficiency capillary electrophoresis and electrospray ionizationmass spectrometry. Anal Chem 2008, 80 (18), 6881-6887. For additionalinformation that may be useful for some designs, see also, Xue Q, ForetF, Dunayevskiy Y M, Zavracky P M, McGruer N E & Karger B L (1997),Multichannel Microchip Electrospray Mass Spectrometry. Anal Chem 69,426-430, Ramsey R S & Ramsey J M (1997), Generating Electrospray fromMicrochip Devices Using Electroosmotic Pumping. Anal Chem 69, 1174-1178,Chambers A G, Mellors J S, Henley W H & Ramsey J M (2011), MonolithicIntegration of Two-Dimensional Liquid Chromatography—CapillaryElectrophoresis and Electrospray Ionization on a Microfluidic Device.Analytical Chemistry 83, 842-849. The contents of these documents arehereby incorporated by reference as if recited in full herein.

FIG. 3A also illustrates that the separation channel 12 can be aserpentine or curvilinear flow channel 12 s and that the at least oneemitter 15 is on a tip of the chip 10 c. However, straight, angled orother configurations of the channel 12 may be used and the emitter(s) 15can be in other locations.

FIGS. 5A-5C illustrate an example of a device 10 with a plurality ofseparation channels 12 and pump channels 20. A plurality of emitters 15are also shown. However, it is also contemplated that each channel 12can communicate with a common emitter as well (not shown). The device 10can be operated so that each emitter concurrently or serially discharges(sprays) the analyte A. FIGS. 5B and 5C illustrate two stackedsubstrates 11 that form the enclosed channels 12, 20. The electricalinputs 116, 120 can be applied using a switch from a common power supplyor each may have a dedicated power input (not shown).

FIGS. 4A-4C are schematic illustrations of capillary devices 10′ usingelectrophoretic-mobility based sample clean-up/infusion ESI. As shown,the devices 10′ can employ a porous tip emitter 15 t capillary setup,but they could be applied with other CE-MS type emitters/setup (e.g.,concentric sheath flow, microchip and the like). As also shown, thedevices 10′ include a capillary separation channel 12′ and a BGE channel22′, each connected to a HV1, HV2 source and meeting at a junction Jproximate the emitter 15 t similar to the embodiments discussed above.However, instead of on an EO pump, capillary driven flow is used. Again,the channel surfaces can be configured to have the desired polarity(charge) and EOF mobility characteristics.

FIG. 4A illustrates that the separation channel 12′ and the BGE channel22′ can each have a positive charge “P” that can be generated by acoating 80 and with a channel 12′ with a reduced mobility EOF coating 88to provide a reduced EOF relative to channel 22′. The device 10′ canoperate with HV1 having a greater positive voltage than HV2.

FIG. 4B illustrates that the device 10′ can be configured with anegative charge “N” and the substrate forming the surfaces of thechannels 12′, 22′ can have a negative charge as discussed with respectto FIG. 2 above. The channel 12′ can also include coating 88. The device10′ can operate with HV1 having a lower positive voltage than HV2.

FIG. 4C illustrates that the capillary system 10′ can also include avacuum 65 applied to the capillary input. The HV1 can have greaterpositive voltage than the HV2 and the vacuum can be operated tocounterbalance EOF. No special coatings are required and the capillariesforming the channels 12′, 22′ can be uncoated.

As discussed above, in some embodiments, the channels 12, 22 and 12′,22′ can be configured with a material or materials (substrate and/orcoating) to have a desired polarity or charge. In some embodiments, thechannels 12, 22 and 12′, 22′ can be configured with a coating to providethe desired polarity or charge. A coating can be dynamic or static.Dynamic coatings generally do not adhere strongly to the channel walland to maintain a stable dynamic coating, dynamic coating reagents areadded to the background electrolyte, thus allowing the coating to becontinuously regenerated. Exemplary dynamic coatings include, but arenot limited to, neutral polymer coatings (e.g., hydroxypropylmethylcellulose coatings) and EOTrol™ coatings commercially availablefrom Target Discovery of Palo Alto, Calif. Static coatings can becovalently bound to the channel wall or strongly adhered to the channelwall by noncovalent interactions (e.g., electrostatic interactions).Exemplary covalent static coatings include, but are not limited to,coatings formed using silane and/or silane reagents such asaminopropyltriethoxysilane, mercaptopropyltrimethoxysilane, polyethyleneglycol (PEG) silane. Silane and/or silane reagents can be used tofunctionalize the surface of the channel wall and/or can act as a baselayer for further modification(s). Exemplary noncovalent static coatingsinclude, but are not limited, coatings comprising polymers includingionic polymers, ionic polymer multilayers and/or polyelectrolytes suchas polyamines (e.g., PolyE-323 a polyamine that interacts with surfacesilanols by electrostatic and hydrogen bonding forces and yields astable positive surface charge over a wide pH range), and polybrenepolymers (e.g., polybrene—poly(vinyl sulfonic acid) polymers andpolybrene—dextran sulfate—polybrene polymers). Other polymer coatingsmay be used such as cross-linked poly(vinyl alcohol).

For positive charge channels 12, 22, suitable coatings 80 include, forexample, but are not limited to, aminopropyltriethoxysilane coatings,PolyE-323 coatings, and Polybrene—dextran sulfate—polybrene coatings.

For negative charge channels 12, 22 glass substrate channels may beused. Glass substrates do not require additional coatings, but may becoated to provide greater or more stable negative charge. However, ifother substrates are used, aluminosilcate coatings or other negativecharge coatings can be used including, for example,mercaptopropyltrimethoxysilane coatings and Polybrene—poly(vinylsulfonic acid) coatings.

The separation channel 12, 12′ (but not the BGE or pump channel 22, 22′)can comprise a coating 88 selected to lower the anodic or cathodic basedEOF in the separation channel 12, 12′ relative to the correspondinganodic or cathodic EOF in the BGE or pump channel 22, 22′. This EOFdifferential can be in any suitable amount and can vary depending on thetarget analyte, the sample under analysis, the degree of sample cleaningdesired, the length of the channels and the like. In some particularembodiments, the separation channel 12, 12′ can have at least a 20%lower EOF (in mobility units E-4 cm²/Vs) than the BGE or pump channel22, 22′. In some embodiments, the EOF in the separation channel 12, 12′can be about 5× to at least 10× lower than the EOF in the other channel22, 22′.

The EOF reduction coating 88 may be an overcoat on an underlyingmonolayer of a material providing a desired polarity, e.g., on a firstionic monolayer coating or placed directly on a channel substrate. Thecoating can be covalently bonded to an underlying coating or substrate.The coating can be non-covalently bonded to an underlying coating orsubstrate. The EOF reduction can be provided as a securely attachedmaterial layer and configured to provide a viscous or flow retardantsurface. In some particular embodiments, the anodic or cathodic EOF inthe separation channel 12 can be about 1E-04 cm²/Vs or less.

The EOF reduction coating 88 can comprise poly(ethylene glycol) (PEG),which may be covalently attached to the first coating on the separationchannel or directly on the separation channel substrate. In someembodiments, this PEGylation can lower the anodic EOF in the separationchannel 12 from about 10E-04 cm²/Vs for an unmodified first coating(APS) surface (such as may be in the BGE or pump channel 22), to lessthan about 1E-04 cm²/Vs.

FIG. 6 illustrates a system 50 with a mass spectrometer 75 with an inletorifice 75 i in communication with at least one fluidic device 10, 10′.The mass spectrometer 75 can generate spectra using a plume sprayed fromone or more emitters 15. In some embodiments, the microchip 10, 10′ canbe configured to spray both an analyte and a reference material from aseparate ESI emitter (not shown). FIG. 6 also illustrates that ananalyzer system 50 that may include a control circuit 90 incommunication with the mass spectrometer 75 and fluidic device 10, 10′and at least one power supply 81 in communication with the electricalinputs 116, 120 on the fluidic device 10, 10′. The control circuit 90can synchronize fluid transport through the fluidic device 10, 10′ withdata acquisition by the mass spectrometer 75. The control circuit 90 maycause different emitters (where used) to sequentially spray or sprayconcurrently.

In some embodiments, the devices 10, 10′ can be used for deposition ontoa target receiving substrate for subsequent analysis. For example, thedevices 10, 10′ can spray or emit fluid from one or more ESI emittersonto a planar substrate for subsequent analysis by Matrix-assisted laserdesorption/ionization (MALDI). See, e.g., Morozov V N (2010)Electrospray Deposition of Biomolecules. In Nano/Micro Biotechnology,pp. 115-162 and Hensel R R, King R C & Owens K G (1997), Electrospraysample preparation for improved quantitation in matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry, RapidCommunications in Mass Spectrometry 11, 1785-1793. MALDI is one of thetwo “soft” ionization techniques besides electrospray ionization (ESI)that allow for the sensitive detection of large, non-volatile and labilemolecules by mass spectrometry. MALDI has developed into anindispensable tool in analytical chemistry, and in analyticalbiochemistry in particular.

FIG. 7 illustrates exemplary operations that can be used to carry outembodiments of the invention. Differential axial transport betweenanalytes of interest and contaminants can be generated in a fluidicdevice having at least one fluidic sample separation flow channel and atleast one ESI emitter in communication with the at least one sampleseparation flow channel (block 100). In response to the generateddifferential axial transport, at least one target analyte contained inthe sample reservoir is selectively transported out the ESI emitter,while inhibiting transport of contaminant materials contained in thesample reservoir to the ESI emitter thereby preferentially directinganalyte molecules out the ESI emitter (block 110).

In some embodiments, the differential axial transport can be generatedby applying a first voltage proximate an ingress end portion of theseparation channel (block 102) and concurrently applying a seconddifferent voltage or ground proximate an ingress end portion of thebackground electrolyte (BGE) channel (block 104). Axial electroosmoticflow (EOF) is generated in the separation and BGE channels in responseto the application of the voltage/electric field so that the EOF in theBGE channel has a greater mobility than that in the separation channel(block 106).

The target analyte can be a biomolecule such as an intact protein (block108).

Embodiments of the invention allow dirty samples to be analyzed byelectrospray mass spectrometry with integrated online sample processing(e.g., clean-up) with relatively non-complex instrumentation. The samplecan be substantially continuously infused for at least 30 seconds,typically between 1 minute to about 2 hours or even longer, moretypically between about 2-60 minutes. The composition of the backgroundelectrolyte can be varied without retention/elution of the sample from achromatographic bed associated with online desalting methods that use aliquid chromatography column coupled to an electrospray interface.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLES Example 1

To evaluate the performance of a prototype device, a conventionalsyringe pump infusion system (FIG. 8) was used to perform ESI-MS of twointact monoclonal antibody (mAb) samples. The transfer capillary wasfused silica with a 100 μm inner diameter. The capillary spray tip had atapered tip with a 10 μm inner diameter. The ESI voltage was applied tothe stainless steel union that connected the ESI emitter to the transfercapillary.

The first sample was purified by washing with background electrolyte(BGE) three times on a 10 kDa molecular weight cutoff (MWCO) filter. TheBGE was a mixture of water with 50% acetonitrile and 0.1% formic acid byvolume. After washing, the sample was diluted to 4 mg/mL with BGE. Thesecond sample was prepared by diluting the raw (20 mg/mL) formulation toa concentration of 2 mg/mL with BGE. Each sample was infused through thesystem for about 15 minutes. A summation of about 5 minutes of ESI-MSdata was used to generate a mass spectrum for each sample.

FIG. 9 shows that the MWCO purified sample yielded a convoluted proteinenvelope centered near 3000 m/z along with a significant amount ofsurfactant peaks between 500 and 1500 m/z. Analysis of this spectrumwould yield only a rough estimate of the average molecular weight of themAb. The sample diluted from the raw formulation yielded anuninterpretable broad peak in the baseline where the mAb charge stateenvelope should be, along with a tremendous amount of background signalranging up to about 2000 m/z. The bottom spectrum (FIG. 9B) was obtainedusing the mAb sample washed three times on a 10 kDa MWCO filter. The topspectrum (FIG. 9A) was obtained using the mAb sample diluted from theraw formulation.

The same two samples run on the syringe pump system were also run on aprototype microfluidic system such as the one shown in FIG. 3A. Thedevice 10 was fabricated in glass substrates using previously reportedmethods. See, Mellors, J. S.; Gorbounov, V.; Ramsey, R. S.; Ramsey, J.M., Fully integrated glass microfluidic device for performinghigh-efficiency capillary electrophoresis and electrospray ionizationmass spectrometry. Anal Chem 2008, 80 (18), 6881-6887. All surfaces ofthe device were covalently modified with an aminopropyl silane (APS)reagent. This results in a strong positive charge on all of the channelsurfaces and therefore a strong anodic EOF. All channels except the EOpump channel were then modified by covalently attaching poly(ethyleneglycol) (PEG). This PEGylation lowered the anodic EOF from approximately10E-04 cm²/Vs for an unmodified APS surface, to less than 1E-04 cm²/Vs.

The channels 12, 22 of the microfluidic device 10 were first filled withBGE (50% acetonitrile, 0.1% formic acid) then one of the samples wasloaded into the sample reservoir 16 (FIG. 3A). When +13 kV was appliedto the sample reservoir 16 and +2 kV was applied to the EO pumpreservoir 20 a stable electrospray plume was generated. After about 2minutes a clean MS spectrum was observed. The ESI-MS signal was verystable for over 15 minutes. Again, about a 5 minute summation was usedto generate a mass spectrum for each of the samples. FIG. 10 shows thatboth samples yielded clean spectra with charge state envelopes centeredaround 2800 m/z. No surfactant ions were observed for either sample.Analysis of either of these spectra would yield relatively accuratemolecular weight estimates of several mAb variants present in thesample. The bottom spectrum (FIG. 10B) was obtained using the mAb samplewashed three times on a 10 kDa MWCO filter. The top spectrum (FIG. 10A)was obtained using the mAb sample diluted from the raw formulation.

These results clearly demonstrate that online sample processing usingdifferences in electrophoretic mobility is an effective method forgenerating high quality ESI-MS data using dirty samples. This methodcould be applied to a number of different applications.

Example 2

To produce the desired EOF, which can differ depending on theapplication, one or more coatings may be used in the prototype device 10described in Example 1 or other suitable fluidic device configurationsto modify one or more surfaces of the device. For example, one or moreof the following coatings may be used to modify one or more surfaces ofthe device 10, 10′. A hydroxypropyl methylcellulose coating may be usedto reduce the EOF. An EOTrol™ coating may be used to optimize the EOF byproviding a coating that is nearly pH- and buffer-independent. Anaminopropyltriethoxysilane coating may be used to provide a strongpositive charge over a wide pH range and/or to provide a base layer foradditional surface modification (e.g., bonding of NHS-PEG).

A mercaptopropyltrimethoxysilane coating may be used to provide a strongnegative charge over a wide pH range and/or as a base layer foradditional surface modification. A PEG silane coating may be used fordirect attachment of PEG to surface silanols without a base layer. APolyE-323 coating may be used to provide a stable positive surfacecharge over a wide pH range. A polybrene—poly(vinyl sulfonic acid)coating, which comprises a double layer where cationic polybreneinteracts electrostatically with surface silanols and anionic poly(vinylsulfonic acid) interacts electrostatically with polybrene, may be usedto provide a stable negatively charged surface. A polybrene—dextransulfate—polybrene coating, which comprises a triple layer coating, maybe used to provide a positively charged surface. A cross-linkedpoly(vinyl alcohol) coating may be used to provide a neutral hydrophilicsurface.

Alternatively and/or in addition to, pressure could be applied to thesample reservoir 16 to produce the desired EOF. For example, a positiveor negative (vacuum) pressure could be applied to the sample reservoir16 to increase or decrease the EOF. In such a system a pressureregulator could be used to “tune” the flow to a level that allows onlythe analyte(s) of interest to reach the ESI interface.

Example 3

FIGS. 11-16 are graphs of mAB signal data. FIGS. 12-14 are graphscomparing syringe pump spectra to a microchip using differential axialtransport for microchip infusion ESI.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof. Although a few exemplary embodiments ofthis invention have been described, those skilled in the art willreadily appreciate that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this invention. Accordingly, all such modifications areintended to be included within the scope of this invention as defined inthe claims. The invention is defined by the following claims, withequivalents of the claims to be included therein.

That which is claimed:
 1. A method of online sample processing forelectrospray ionization (ESI), comprising: generating differential axialtransport in a fluidic device having at least one fluidic sampleseparation channel and at least one ESI emitter in communication withthe at least one sample separation channel; and in response to thegenerated differential axial transport, selectively transporting atleast one target analyte contained in a sample reservoir incommunication with the sample separation channel to the at least one ESIemitter thereby inhibiting transport of contaminant materials containedin the sample reservoir.